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First published online September 28, 2006
doi: 10.1242/10.1242/dev.02581
1 Department of Biochemistry and Biophysics and Programs in Developmental
Biology, Genetics and Human Genetics, Cardiovascular Research Institute,
University of California, San Francisco, San Francisco, CA 94143-2711,
USA.
2 Division of Biological Science, Graduate School of Science, Nagoya University,
Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.
3 Department of Biological Sciences, Graduate School of Science, University of
Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan.
* Authors for correspondence (e-mail: htakeda{at}biol.s.u-tokyo.ac.jp; didier_stainier{at}biochem.ucsf.edu)
Accepted 10 August 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Myocardial migration, mtx1 (mxtx1), Yolk syncytial layer (YSL), fibronectin, natter, Zebrafish
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
de Souza and Niehrs, 2000). In
addition, reciprocal cell-cell interactions between the extra-embryonic
ectoderm and the epiblast play crucial roles in early anterior-posterior
patterning and mesoderm induction during mouse embryogenesis
(Brennan et al., 2001).
Similarly, in zebrafish, the extra-embryonic yolk syncytial layer (YSL) is
known to function as an important signaling center for mesoderm induction and
early dorsoventral patterning (Mizuno et
al., 1996; Chen and Kimelman,
2000) (reviewed by Sakaguchi
et al., 2002). However, little is known about the roles the
extra-embryonic tissues might play in later developmental events, such as
organogenesis.
The heart is the first organ to form and function during vertebrate
development. In all vertebrates, the heart tube develops from bilateral
populations of precursor cells in the anterior lateral plate mesoderm
(reviewed by McFadden and Olson, 2002). In zebrafish, these myocardial
precursors migrate in between the pharyngeal endoderm and the extra-embryonic
YSL toward the midline and subsequently fuse to form the functional heart tube
(reviewed by Stainier, 2001;
Trinh and Stainier, 2004).
Large-scale forward genetic screens in zebrafish have identified mutations in
eight loci that disrupt this migration process, resulting in the formation of
two separate hearts, a phenotype referred to as cardia bifida
(Chen et al., 1996;
Stainier et al., 1996;
Alexander et al., 1998).
Previous analyses have demonstrated that the endodermal layer is essential for
myocardial migration, as all mutants that lack pharyngeal endoderm show cardia
bifida (Alexander et al., 1999;
Reiter et al., 1999;
Kikuchi et al., 2000). Cell
transplantation analyses have further shown that wild-type endoderm can rescue
the myocardial migration defects in the casanova/sox32 mutant
(David and Rosa, 2001),
further supporting the idea that the pharyngeal endoderm is essential for
myocardial migration. Cellular analyses have indicated that the epithelial
integrity of the myocardial precursors is also important for their migration,
as evidenced by the cardia bifida mutation natter, which affects
fibronectin 1 and appears to disrupt the polarity of the myocardial
precursors (Trinh and Stainier,
2004). This finding is consistent with previous observations in
mouse and chick embryos that Fibronectin, a major constituent of the
extracellular matrix (ECM), is required for heart tube formation
(Linask and Lash, 1988;
George et al., 1993). The
mechanisms that lead to cardia bifida in the miles apart mutant are
still unclear, although the mutant exhibits dysmorphic pharyngeal endoderm
(Kupperman et al., 2000). The
importance of the pharyngeal endoderm in myocardial migration has been well
documented; however, a role for the extra-embryonic YSL in this process has
not been identified from the forward genetic analyses.
The YSL is the cortical cytoplasm region of the yolk cell, and it contains
hundreds of nuclei (Trinkaus,
1992). The YSL is formed at the 500- to 1000-cell stage as
marginal blastomeres collapse onto the yolk cell
(Kimmel and Law, 1985).
Although the YSL usually persists up to 9 days post-fertilization, it does not
contribute cells or nuclei to any adult tissues
(D'Amico and Cooper, 2001).
Therefore, once established at the blastula stage, the YSL takes an
exclusively extra-embryonic cell fate.
Several zebrafish mix-family genes, such as bonnie and
clyde (bon), mtx1 (mxtx1 - Zebrafish
Information Network), and mtx2 (mxtx2 - Zebrafish
Information Network), are expressed in the YSL
(Alexander et al., 1999;
Hirata et al., 2000;
Kikuchi et al., 2000).
bon and mtx2 are expressed in the marginal mesendodermal
cells and the YSL, while mtx1 is expressed exclusively in the YSL
during blastula and gastrula stages
(Alexander et al., 1999;
Hirata et al., 2000). In
higher vertebrates, mix-family genes such as mixer and
Mixl1 play important roles in endoderm and axial mesoderm development
(Henry and Melton, 1998;
Hart et al., 2002). In
zebrafish, bon plays a crucial role in early endoderm differentiation
(Kikuchi et al., 2000), and
mtx2 appears to regulate epiboly movements
(Bruce et al., 2005). However,
the function of mtx1 remains unexplored.
In this study, we show that the YSL-specific gene mtx1 is required for myocardial migration. We also show that mtx1 regulates natter/fibronectin 1 expression, and that mtx1 and natter/fibronectin 1 interact genetically. Based on these and other data, we propose that the extra-embryonic YSL regulates cardiac morphogenesis, and does so in part by regulating ECM assembly in embryonic tissues.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Injection of morpholino antisense oligonucleotides and synthesized mRNA into the YSL
Three independent antisense morpholinos (MOs) for the mtx1 gene
(Gene Tools, LLC) were injected into the YSL at the 1000-cell stage (3 hpf) as
previously described (Sakaguchi et al.,
2001). Eight nanograms/embryo of mtx1 MO(A)
(5'-CATCAATAGTGCTGTCTTTCCACAT-3'), 4 ng/embryo of mtx1
MO(B) (5'-GCGTCTTCACTGGTGGAATCCTGGA-3'), 8 ng/embryo of
mtx1 MO(C) (5'-TAAACATGACAGCCCACCTGTATGC-3'), and 8
ng/embryo of 4 bp mismatch control mtx1 MO(A)
(5'-CAaCAAaAGTGCTGTCTaTCCtCAT-3') were injected into the YSL. Two
nanograms/embryo of laminin c1 MO
(5'-TGTGTCCTTTTGCTATTGCGACCTC-3')
(Parsons et al., 2002) was
injected into 1-cell stage embryos.
Capped sense mtx1 mRNAs were synthesized using the mMessage
mMachine in vitro transcription kit (Ambion), and purified as previously
described (Sakaguchi et al.,
2001). One hundred picograms/embryo of mtx1 mRNA was
injected into the YSL at the 1000-cell (3 hpf) or dome (4.3 hpf) stage.
RT-PCR and genotyping of natter embryos
For RT-PCR of mtx1 cDNA, wild-type embryos and mtx1 MO(C)
injected embryos were collected at shield stage and total RNA was isolated. RT
reactions were performed using the SuperScriptII RT-PCR kit (Gibco BRL) with
the mtx1 specific RT primer
(5'-TCCTCAGTGGAGTGGTTATTTAGTC-3') according to the manufacturer's
recommendations. PCR was performed using the primer pair
(5'-GAGAGTCTTTCCCAAACCACAG-3' and
5'-CATATTGTTGTAGGCTGGCAAGT-3') to amplify mtx1 cDNA
fragments.
Genotyping by RFLP of nattertl43c was performed by PCR
as previously described (Trinh and
Stainier, 2004).
In situ hybridization and immunohistochemistry
Whole-mount in situ hybridizations were performed as described
(Sakaguchi et al., 2001).
Immunohistochemistry was performed as described
(Trinh and Stainier, 2004). We
used the following antibodies: rabbit polyclonal anti-Fibronectin (Sigma)
(Trinh and Stainier, 2004) at
1:200; mouse IgG1 anti-ß-catenin (Sigma) at 1:500; rabbit
polyclonal anti-Laminin 1 (Sigma) at 1:200. For whole-mount anti-Laminin 1
staining, we used the ABC staining kit (Vector Laboratory) after treating with
an anti-rabbit IgG biotin-conjugated antibody. Fluorescence confocal images
were acquired using a Zeiss LSM5 Pascal confocal microscope.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), we
screened for genes that are expressed in the YSL at the late blastula stage
and examined their function by MO
(Heasman, 2002) knockdown
experiments. We found that an MO designed to knock down the 124H4
clone caused heart malformations and subsequently determined that the
124H4 clone is identical to the previously reported transcription
factor gene mtx1 (Hirata et al.,
2000). mtx1 is a Mix-type homeobox gene that is expressed
exclusively in the YSL from the oblong stage (3.7 hpf) until 80% epiboly (8.3
hpf) (Hirata et al., 2000)
(data not shown). No mtx1 transcripts can be detected after gastrula
stage, or in any embryonic tissues (Hirata
et al., 2000). However, it remains theoretically possible that low
levels of mtx1 transcripts are present in embryonic tissues. In all
experiments, we injected mtx1 MOs into the YSL at the 1000-cell stage
(3 hpf) to knock down mtx1 function in this tissue. We found that
mtx1 MO(A) injected embryos displayed pericardial edema
(Fig. 1B, arrowhead) and a
flattened hindbrain (Fig. 1B,
arrow) at 34 hpf. Injecting a 4 bp mismatch control MO into the YSL did not
cause any phenotype (Fig. 1A).
Interestingly, when we investigated the cause of the pericardial edema, we
found that approximately 30% (14/51) of mtx1 MO(A)-injected embryos
showed cardia bifida at 34 hpf (Fig.
1D, arrowheads). Co-injection of Rhodamine Dextran with
mtx1 MO(A) allowed us to visualize the distribution of the MO and
showed it to be mostly restricted to the YSL even as late as 34 hpf
(Fig. 1E,F). These results
indicate that the YSL regulates myocardial migration.
We further analyzed the cardia bifida phenotype of mtx1
MO(A)-injected embryos by examining the expression of the myocardial-specific
marker cmlc2 (myl7 - Zebrafish Information Network)
(Yelon et al., 1999). We first
examined myocardial migration at the 21-somite stage (19.5 hpf), a time at
which the two groups of myocardial precursors initially fuse at the midline
(Yelon et al., 1999). We found
that 100% (14/14) of mtx1 MO(A)-injected embryos exhibited two
bilateral clusters of myocardial cells at this stage, indicating a failure in
their migration to the midline (Fig.
1H). By contrast, none of the 4 bp mismatch control MO-injected
embryos exhibited a myocardial migration defect
(Fig. 1G). Time-lapse
observations of myocardial migration with Tg(cmlc2:GFP) embryos
(Huang et al., 2003) indicated
that the myocardial precursors, which failed to fuse at the 21-somite stage,
had reached the midline by 24 hpf in 4/6 of mtx1 MO(A)-injected
embryos (Fig. 1N), while in the
other two cases they remained bilateral
(Fig. 1O). The embryos showing
delayed myocardial fusion sometimes established circulation by 34 hpf, whereas
the embryos showing bilateral myocardial precursors at 24 hpf developed clear
cardia bifida by 34 hpf. Consistent with these observations, we found that at
38 hpf the cmlc2-expressing myocardial precursors remained bilateral
in approximately 30% (4/11) of mtx1 MO(A)-injected embryos
(Fig. 1Q). These results
indicate that the migration of the myocardial precursors was severely
compromised in mtx1 MO(A)-injected embryos.
Specificity of mtx1 morpholinos
To test the specificity of MO(A) for mtx1, we designed and
injected two additional mtx1 MOs [MO(B) and (C)] into the YSL. Both
MO(B) and MO(C) caused the same phenotype as MO(A), and myocardial cells
failed to fuse at the midline at the 21-somite stage in all injected embryos
(16/16, 18/18, respectively; Fig.
1I,J), suggesting that the mtx1 MOs function in a
target-specific manner.
As cytoplasmic bridges connect the yolk cell to marginal blastomeres until
sphere stage (4 hpf) (Cooper and D'Amico,
1996), MOs injected into the YSL before sphere stage can diffuse
into embryonic tissues. As it remained formally possible that mtx1
was expressed at very low levels in marginal blastomeres, and to assess the
specificity of the MOs, we wanted to test whether restoring mtx1 mRNA
specifically in the YSL would rescue the myocardial migration phenotype of
mtx1 MO-injected embryos. We injected mtx1 MO(C) into the
YSL at the 1000-cell stage (3 hpf) and subsequently injected some of these
embryos with mtx1 mRNA into the YSL at dome stage (4.3 hpf). While
all (n=8) of the embryos injected with mtx1 MO(C) showed
delayed myocardial migration at the 21-somite stage
(Fig. 1K), 50% (3/6) of the
embryos subsequently injected with mtx1 mRNA into the YSL exhibited
wild-type-like myocardial migration (Fig.
1L). These data indicate that mtx1 functions in the YSL
to regulate myocardial migration.
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), we first asked whether endoderm development in
mtx1 MO-injected embryos was affected. Expression of early endoderm
markers, such as sox17 (Alexander
and Stainier, 1999) and casanova/sox32
(Dickmeis et al., 2001;
Kikuchi et al., 2001;
Sakaguchi et al., 2001),
appeared unaffected in mtx1 MO(A)-injected embryos
(Fig. 3A-D), indicating that
the loss of mtx1 function does not affect early endoderm formation.
Later in development, myocardial cells migrate underneath a sheet of
pharyngeal endoderm toward the midline
(Trinh and Stainier, 2004).
Therefore, we examined pharyngeal endoderm development in mtx1
MO(A)-injected embryos and it appeared unaffected, as assessed by examining
the pharyngeal endoderm-specific GFP expression in
Tg(her5:GFP)ne206 embryos
(Tallafuss and Bally-Cuif,
2003) at the 18-somite stage
(Fig. 3E,F). Despite the
absence of obvious pharyngeal endoderm defects, these embryos exhibited
myocardial migration defects at the 21-somite stage (data not shown),
indicating that mtx1 had been knocked down in these embryos. We also
examined Tg(her5:GFP)ne2067; Tg(cmlc2:GFP) embryos in
cross-sections and did not detect any obvious defects in her5:GFP
expression in mtx1 MO-injected embryos at the level of the cardiac
mesoderm at the 17-somite stage (17.5 hpf)
(Fig. 3G,H). These data suggest
that the YSL regulates myocardial migration independently of pharyngeal
endoderm formation.
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;
Ober et al., 2003). We were
able to visualize these condensing foregut endodermal cells at the 15-somite
stage with a sox17-promoter-GFP transgenic line,
Tg(sox17:GFP)s870 (Y.K. and W. Chung, unpublished)
(Fig. 3I). In mtx1
MO(A)-injected embryos at the 15-somite stage, foregut endodermal cell
migration appeared to be delayed, as evidenced by its sheet-like appearance
(Fig. 3J). By 24 hpf, foregut
endodermal cells, marked by foxa1 expression, have already formed a
rod in control embryos (Fig.
3K), whereas this cell migration was delayed in mtx1
MO(A)-injected embryos (12/15; Fig.
3L). Due to this cell migration delay, approximately 30% (6/18) of
mtx1 MO(A)-injected embryos developed duplicated hepatic and anterior
pancreatic buds, as assessed by Tg(gutGFP)s854
(Field et al., 2003;
Ober et al., 2003) expression
at 54 hpf (Fig. 3M,N). As
endodermal cells migrate on the YSL to coalesce at the midline, the YSL is
probably also regulating this process.
natter/fibronectin 1 is regulated by mtx1
To investigate the mechanisms by which the YSL regulates myocardial
migration, we next focused on the natter/fibronectin 1 gene, which,
like mtx1, is necessary for myocardial migration but appears to act
independently from the endoderm (Trinh and
Stainier, 2004). In addition, mtx1 MO-injected embryos
exhibited similar phenotypes to those of natter/fibronectin 1
mutants, such as a flattened hindbrain and a partially penetrant cardia bifida
(Fig. 1B,
Fig. 5C)
(Trinh and Stainier, 2004). We
analyzed natter/fibronectin 1 expression in mtx1
MO(A)-injected embryos at several stages and detected a clear downregulation
at the 14-somite stage (16 hpf) in the anterior lateral plate mesoderm, when
the myocardial precursors start to migrate to the midline
(Fig. 4A,B). This
downregulation of natter/fibronectin 1 expression was also detected
at the 21-somite stage (19.5 hpf), when the myocardial precursors first fuse
at the midline (Fig. 4C,D). At
the 21-somite stage, natter/fibronectin 1 is expressed in the
laterally located myocardial cells and medially located endocardial cells
(Trinh and Stainier, 2004). We
observed a downregulation of natter/fibronectin 1 expression in both
cell populations in mtx1 MO(A)-injected embryos
(Fig. 4D). We also examined
Fibronectin protein deposition in mtx1 MO(A)-injected embryos. In
wild-type embryos at the 18-somite stage, Fibronectin is deposited on the
basal side of the myocardial precursors as well as on the basal side of the
pharyngeal endoderm (Fig. 4I)
(Trinh and Stainier, 2004). In
mtx1 MO(A)-injected embryos, Fibronectin deposition was greatly
reduced around both the myocardial precursors and the pharyngeal endoderm
(Fig. 4J).
As loss of mtx1 function downregulated natter/fibronectin 1 gene expression, we next asked whether mtx1 overexpression could alter natter/fibronectin 1 expression. Initially, we injected 100 pg mtx1 mRNA into 1-cell-stage embryos; however, these injected embryos showed severe gastrulation and epiboly defects. We then injected the same amount of mtx1 mRNA into the YSL at the 1000-cell stage to overexpress mtx1 preferentially in this tissue. We found that all these injected embryos appeared morphologically unaffected at 34 hpf (data not shown). However, interestingly, we found that natter/fibronectin 1 expression in the anterior lateral plate mesoderm of these embryos appeared to be upregulated at the 18-somite stage (Fig. 4E-H). Fibronectin immunoreactivity was also clearly increased in embryonic tissues (Fig. 4K). These data suggest that the extra-embryonic YSL is regulating embryonic natter/fibronectin 1 expression in adjacent embryonic tissues.
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Homozygous natter mutant embryos injected with mtx1 MO(A) frequently failed to complete epiboly and died after yolk rupture during gastrulation (data not shown). The embryos that survived appeared similar to mtx1 MO(A)-injected heterozygous natter embryos at 34 hpf (Fig. 5D).
Morphogenesis of mtx1 MO-injected heterozygous natter embryos
As mtx1 MO-injected heterozygous natter embryos showed
severe morphogenetic defects, we carried out a time-course analysis to
determine how these defects developed during embryogenesis
(Fig. 6). mtx1
MO-injected heterozygous natter embryos appeared normal at the
tailbud stage (Fig. 6A,F; 10
hpf). However, 2 hours later, at the 6-somite stage, the YSL at the hindbrain
level started to darken (Fig.
6B,G; 12 hpf). By the 17-somite stage (17.5 hpf), the head
structure appeared to be collapsing onto the YSL in mtx1 MO-injected
heterozygous natter embryos (Fig.
6C-D,H-I). At 24 hpf, somite boundaries became hard to
distinguish, especially in the anterior part
(Fig. 6E,J). This result is
consistent with previous observations that natter/fibronectin 1 and
fibronectin 3 are required for somite formation
(Julich et al., 2005;
Koshida et al., 2005). By 34
hpf, the hindbrain was completely collapsed onto the yolk ball and most somite
boundaries appeared to be missing (Fig.
5B). These data show that major defects appeared at late
segmentation stages in mtx1 MO-injected heterozygous natter
embryos.
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), the
rhombomeres 3 and 5 marker krox20 (egr2b - Zebrafish
Information Network) (Fig.
7A,B) (Oxtoby et al., 1993), and the adaxial cell and segmented
somite marker myod (Fig.
7C,D) (Weinberg et al.,
1996) appeared in a properly segmented manner, indicating that
these embryos were being patterned properly. Also, although myocardial cells
failed to migrate in these embryos, they continued to express the myocardial
marker cmlc2 (Fig.
7E,F).
To further understand why mtx1 MO-injected heterozygous
natter embryos could not maintain their morphology, we examined ECM
assembly by analyzing Laminin 1 immunoreactivity
(Parsons et al., 2002;
Pollard et al., 2006). We
found that mtx1 MO-injected heterozygous natter embryos
exhibited greatly reduced Laminin 1 immunoreactivity in the hindbrain at 24
hpf, while it appeared unchanged in their trunk and tail
(Fig. 7H,J). Confocal images of
cross-sections at the level of the cardiac mesoderm further illustrated the
fact that Laminin 1 immunoreactivity was reduced in mtx1 MO-injected
heterozygous natter embryos (Fig.
7L). As mtx1 MO injections into wild-type embryos did not
significantly reduce Laminin 1 immunoreactivity (data not shown), these data
suggest that losing one copy of natter/fibronectin 1 in the absence
of mtx1 function leads to a further disruption of ECM assembly.
To further analyze the role of the YSL in ECM assembly, we injected
mtx1 MO(A) into the YSL of embryos previously injected with
laminin c1 (lamc1) MO at the 1-cell stage. lamc1
encodes Laminin 
1 and is essential for notochord differentiation and
Laminin 1 immunoreactivity (reviewed by
Stemple, 2005;
Parsons et al., 2002;
Pollard et al., 2006). Embryos
injected with lamc1 MO at the 1-cell stage exhibited a shortened body
axis and defects in notochord differentiation at 30 hpf
(Fig. 7M)
(Parsons et al., 2002). When
we injected mtx1 MO(A) into the YSL of embryos previously injected
with lamc1 MO, approximately 50% of embryos (47/90) appeared to die
after yolk rupture during segmentation stages. Surviving embryos appeared to
exhibit an enhanced phenotype, in which body structures were collapsing in the
hindbrain region (22/90) (Fig.
7N) or in the entire head region (17/90)
(Fig. 7O) at 30 hpf. These
phenotypes were not seen in lamc1 MO-injected embryos or
mtx1 MO(A)-injected embryos. These results further support the idea
that the YSL influences embryonic ECM assembly.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), loss of Mtx1 function also leads to cardia bifida.
Interestingly, we also found a genetic interaction between mtx1 and
natter/fibronectin 1. Taken together, these data indicate that the
extra-embryonic YSL regulates cardiac morphogenesis, at least in part by the
mtx1-natter/fibronectin 1 pathway.
Specificity of mtx1 MOs
In this study, we injected mtx1 MOs into the YSL at the 1000-cell
stage (3 hpf) to knock down mtx1 function in the YSL. All three
mtx1 MOs caused the same phenotype, as assessed by the expression of
the myocardial marker cmlc2 at the 21-somite stage
(Fig. 1H-J). These data
strongly suggest that mtx1 MOs function in a target-specific manner.
Injection of the mtx1 MO(C) into 1-cell-stage embryos caused cardia
bifida, although at low frequency (data not shown). As the YSL contains
hundreds of nuclei, we suspect that knocking down genes in the YSL requires a
higher concentration of MOs, one achieved by YSL injections.
We propose that our mtx1 MO injections are specifically knocking
down mtx1 function in the YSL for the following reasons: first,
mtx1 appears to be exclusively expressed in the YSL
(Hirata et al., 2000), and we
injected mtx1 MOs into this tissue. Second, the same concentration of
a 4 bp mismatch mtx1 MO(A) did not cause any obvious phenotypes when
injected into the YSL (Fig.
1A,G). Third, the splice donor MO, mtx1 MO(C), leads to a
frame shift and premature translational termination of mtx1
(Fig. 2). Finally, restoring
mtx1 mRNA in the YSL rescued the myocardial migration defect
(Fig. 1L).
Roles of the extra-embryonic YSL in myocardial migration
In zebrafish, myocardial precursors migrate in between the endodermal layer
and the extra-embryonic YSL (Fig.
8A) (Trinh and Stainier,
2004) (reviewed by Stainier,
2001). Previous studies have demonstrated that the endodermal
layer is essential for myocardial migration (reviewed by
Stainier, 2001). This study
clearly illustrates for the first time that the extra-embryonic YSL also plays
important roles in myocardial migration.
As mtx1 is a strictly zygotically expressed gene, a maternal
factor probably controls mtx1 gene expression
(Fig. 8B). mtx2, which
is expressed in the YSL and marginal cells
(Hirata et al., 2000), appears
to be regulated by the maternal factor Eomesodermin
(Bruce et al., 2005). However,
Eomesodermin does not appear to regulate mtx1 gene expression
(Bruce et al., 2005),
indicating that another maternal factor is involved. Interestingly, the
phenotype of mtx1 MO-injected heterozygous natter embryos
appears similar to that of the maternal mutant pollywog
(Fig. 5B)
(Wagner et al., 2004),
suggesting that Pollywog is an upstream regulator of mtx1.
Once mtx1 expression is established in the YSL, it appears to
regulate embryonic natter/fibronectin1 gene expression in a
non-cell-autonomous manner (Fig.
4). It is striking that loss of mtx1 function leads to
downregulation of natter/fibronectin 1 expression and that, by
contrast, gain of mtx1 function leads to upregulation of
natter/fibronectin 1 expression in the anterior lateral plate
mesoderm. However, the natter/fibronectin 1 gene cannot be a direct
target of Mtx1, as their expression patterns do not appear to overlap. No
fibronectin 1 transcripts can be detected at shield stage
(Trinh and Stainier, 2004),
when mtx1 is expressed most strongly
(Hirata et al., 2000). Zygotic
fibronectin 1 expression appears to initiate at 60% epiboly (6.5 hpf)
in the marginal blastomeres but not in the YSL
(Trinh and Stainier, 2004),
while mtx1 appears to be expressed only in the YSL until 80% epiboly
(8.3 hpf) (Hirata et al.,
2000). Therefore, the regulation of natter/fibronectin 1
by Mtx1 is probably indirect. Our data suggest that factors unidentified to
date are regulated by Mtx1 in the YSL and work non-cell-autonomously to
regulate natter/fibronectin 1 expression in the embryonic lateral
plate mesoderm at mid-segmentation stages
(Fig. 7). Further investigation
will be required to identify the molecules that function downstream of
mtx1 and upstream of natter/fibronectin 1. The steroidogenic
enzyme Cyp11a1 (Hsu et al.,
2006) and Rac-GAP Chimerin 1
(Leskow et al., 2006) have
been reported to function in the YSL to regulate epiboly cell movement. It
will be interesting to investigate the role of these proteins in
natter/fibronectin 1 gene expression and myocardial migration.
mtx1 might be working together with casanova/sox32, which
is expressed in the endoderm and YSL and is responsible for endoderm formation
(Dickmeis et al., 2001;
Kikuchi et al., 2001;
Sakaguchi et al., 2001).
Although casanova MO injections into the YSL did not cause any
obvious morphological phenotype (Sakaguchi
et al., 2001), co-injection of mtx1 and casanova
MOs into the YSL dramatically increased the penetrance of cardia bifida at 34
hpf (data not shown). More detailed analysis will be required to further
investigate this potential interaction between casanova and
mtx1.
The idea that mtx1 regulates natter/fibronectin 1 gene expression is also supported by the fact that mtx1 and natter/fibronectin 1 show dosage-sensitive genetic interactions (Fig. 5). The phenotype of mtx1 MO-injected heterozygous natter embryos (Fig. 5B) is much more severe than that of mtx1 MO-injected wild-type embryos (Fig. 1B) or uninjected homozygous natter mutant embryos (Fig. 5C), implying that there is another pathway downstream of mtx1, working in parallel to the mtx1-natter/fibronectin 1 pathway (Fig. 8). Based on our observation that Laminin 1 immunoreactivity is greatly downregulated in mtx1 MO-injected heterozygous natter embryos (Fig. 7G-L), while it is not significantly affected in mtx1 MO-injected wild-type embryos, ECM components other than Fibronectin also appear to be regulated by mtx1 (Fig. 8). This idea is further supported by the observation that embryos injected with both lamc1 and mtx1 MOs showed a much more severe phenotype than that of lamc1 MO-injected embryos (Fig. 7M), mtx1 MO-injected embryos (Fig. 1B), or an addition of these two phenotypes. These data suggest that lacking multiple components of the ECM can lead to synergistic defects. We therefore propose that the extra-embryonic YSL regulates heart organogenesis and body structure maintenance at least in part by controlling ECM assembly (Fig. 8).
The YSL as a scaffold on which many cell types migrate
Although mtx1 MO-injected embryos developed normal pharyngeal
endoderm, they exhibited foregut endoderm migration defects
(Fig. 3). This foregut endoderm
phenotype is not seen in natter mutants
(Trinh and Stainier, 2004),
indicating that foregut endoderm migration is not regulated by the
mtx1-natter/fibronectin 1 pathway. Vegfc has been shown
previously to regulate foregut endoderm migration
(Ober et al., 2004), and it
will be interesting to investigate whether the YSL regulates Vegfc function.
Furthermore, the YSL might be serving as a scaffold for many cell types,
including endodermal cells and myocardial cells, which migrate on it. We
hypothesize that mtx1 might be involved in establishing the YSL as a
scaffold for cell migration, and therefore loss of mtx1 function
would compromise the motility of many cell types that migrate on the YSL
(Fig. 8). This hypothesis is
consistent with the fact that mtx2 in the YSL regulates embryonic
cell movements during epiboly (Bruce et
al., 2005).
Role of extra-embryonic tissues in vertebrate heart development
Several mutations in mouse, such as those in the fibronectin, Gata4,
Mesp1, Furin and Foxp4 genes, are known to affect the migration
of myocardial precursors and lead to cardia bifida
(George et al., 1993;
Kuo et al., 1997;
Molkentin et al., 1997;
Roebroek et al., 1998;
Saga et al., 1999;
Constam and Robertson, 2000;
Li et al., 2004). In mouse
embryos around embryonic day 7.5, myocardial precursors appear to lie adjacent
to the extra-embryonic visceral endoderm
(Kaufman, 1992). This scheme
might be equivalent to that in zebrafish, in which myocardial precursors
migrate on the extra-embryonic YSL. Interestingly, chimeric analyses in mouse
have demonstrated that Gata4 function in the visceral endoderm is required for
myocardial migration (Watt et al.,
2004). Therefore, we speculate that the YSL in zebrafish has a
role in myocardial migration equivalent to that of the visceral endoderm in
mouse. Several genes, such as ferroportin1
(Donovan et al., 2000), are
expressed in the YSL in zebrafish and the visceral endoderm in mouse, further
highlighting the similarities between these two tissues. It will therefore be
interesting to assess the role of the visceral endoderm in establishing ECM
assembly in mouse embryonic tissues.
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ACKNOWLEDGMENTS |
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REFERENCES |
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